Soluble vaccinia virus protein that binds chemokines

Details Soluble vaccinia virus protein that binds chemokines Soluble vaccinia virus protein that binds chemokines

1999-11-22

2002-03-12

Stucker, Jeffrey (Department: 1648)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Virus or component thereof

C424S205100, C435S235100, C435S236000

Reexamination Certificate

active

06355252

ABSTRACT:

This invention relates to a novel chemokine-binding protein and chemokine-binding fragments thereof. In particular, the invention relates to uses of the protein or fragments in a medicament, for example as an anti-viral or an anti-inflammatory agent; and to methods for the detection and inhibition of chemokines.
Chemokines are a family of small secreted polypeptides that regulate trafficking and effector functions of leukocytes, and play an important role in inflammation and host defence against pathogens (D'Souza & Harden, 1996; Fauci, 1996; Howard et al., 1996; Murphy, 1996; Premack & Schall, 1996; Baggiolini et al., 1997). More than 30 chemokines have been identified and these are divided into at least three structural groups based on the number and arrangement of conserved cysteines: CC (&bgr;) chemokines such as RANTES (for regulation-upon-activation, normal T cell expressed and secreted) and macrophage inflammatory protein (MIP)-1&agr;, CXC (&agr;) chemokines such as interleukin-8 (IL-8) and growth-related oncogene (GRO)-&agr;, and the C chemokine lymphotactin. The different chemokines have evolved to function with particular cell types: many CC chemokines are chemoattractants for monocytes or thymocytes, while many but not all CXC chemokines are chemoattractants for neutrophils. However, there is no rule relating chemokine structure and cellular specificity to other leukocyte subtypes. For example, the C chemokine lymphotactin is selective for T cells, the CC chemokine eotaxin is specific for eosinophils and the CXC chemokines IFN-&ggr;-induced protein (IP-10) and monokine induced by IFN-&ggr; (Mig) attract activated T cells.
Chemokine receptors (CKRs) are seven transmembrane domain proteins and are coupled to G proteins for signal transduction. Most CXC chemokines have high affinity for only a single CKR (IL-8 is an exception in binding to CXCR1 and CXCR2), whereas most CC chemokines bind to more than one CKR (Baggiolini et al., 1997).
The activity of chemokines is tightly regulated to prevent excessive inflammation that can cause disease. Inhibition of chemokines by neutralising antibodies in animal models (Sekido et al., 1993) or disruption of mouse chemokine genes (Cook et al., 1995) have confirmed a critical role of chemokines in vivo in inflammation mediated by virus infection or other processes. The production of soluble versions of cytokine receptors containing only the extracellular binding domain, represents a physiological and therapeutic strategy to blockade the activity of some cytokines (Rose-John & Heinrich, 1994). However, the seven transmembrane domain structure of CKRs makes the construction of soluble, inhibitory CKRs difficult, and thus mutated chemokine antagonists, blocking peptides or antibodies are alternative inhibitors of chemokines under evaluation (D'Souza & Harden, 1996; Howard et al., 1996).
CKRs play a critical role in transmission and dissemination of HIV by acting as a cofactor which is required together with CD4 for virus entry and infection (D'Souza & Harden, 1996; Fauci, 1996). The CXCR4 CKR is a cofactor for T cell line-tropic HIV isolates, whereas the CCR5 (and CCR3) CKRs are involved in infection by macrophage-tropic HIV strains. The importance of CCR5 in vivo is supported by the finding that individuals who are homozygous for a mutant version of the CCR5 gene are resistant to HIV infection. Binding of chemokines or mutated chemokine antagonists to CKRs block HIV infection, illustrating the potential of the blockade of HIV-CKR interaction as a preventive and therapeutic strategy against HIV (D'Souza & Harden, 1996; Fauci, 1996).
Poxviruses are a group of large DNA viruses that replicate in the cytoplasm of the cell and encode many of their own enzymes for transcription and DNA replication (Moss, 1996). Vaccinia virus (VV) is the most intensively studied poxvirus and is famous as the live vaccine that was used to eradicate smallpox (Fenner et al., 1988). Since 1982 VV has also been widely used as an expression vector and in vaccine research (Moss, 1991). The genome of VV strain Copenhagen has been completely sequenced and contains approximately 200 genes (Goebel et al., 1990). Those near the centre of the genome are mostly highly conserved between poxviruses and many are essential for virus replication. In contrast, genes located towards either end of the genome are more variable between different viruses and are frequently nonessential for virus replication (Johnson et al., 1993). A subset of these non-essential virus genes are important for blocking specific components of the host immune system and many affect virus virulence. Thus VV and other poxviruses express proteins that are able to block the action of interferons, complement, cytokines, chemokines, inflammation and fever (Alcami & Smith, 1995a; McFadden et al., 1995; Smith, 1996; Spriggs, 1996; Smith et al., 1997c). Many of these proteins are secreted from the infected cell and bind to host factors in solution or at the cell surface. In many cases there is amino acid similarity between the virus protein and the extracellular ligand-binding domain of a cell surface receptor for a particular ligand. In these cases it seems likely that the virus gene has been derived from the host during evolution.
Soluble chemokine-binding proteins have been previously detected in poxviruses. Firstly, the myxoma virus T7 protein, which was first identified as a soluble IFN-&ggr;R (Upton et al., 1992), binds to a range of chemokines through the heparin-binding domain and affects the infiltration of cells into infected tissue (Mossman et al., 1996; Lalani et al., 1997). The protein is described in WO 96/33730, designated CBP-1. In contrast, the IFN-&ggr;R from orthopoxviruses such as VV does not bind chemokines. (Alcami et al., 1998). Secondly, it was demonstrated that VV strain Lister expresses a soluble 35 kDa protein that is secreted from infected cells and which binds many CC chemokines (Graham et al., 1997; Smith et al., 1997a; Smith et al., 1997b; Alcami et al., 1998), but not CXC chemokines, through a domain distinct from the heparin-binding domain (Smith et al., 1997a; Smith et al., 1997b; Alcami et al., 1998). This protein has been called vCKBP (Alcami et al., 1998). The protein is also described in WO 97/11714, as p35. Very similar proteins are made by some but not all VV strains, cowpox virus, the leporipoxviruses, Shope fibroma virus (SFV) and myxoma virus (called T1 protein), and by variola major viruses e.g. protein G5R in India-1967 (Shchelkunov et al., 1994). Another VV gene encodes a protein more distantly related to vCKBP. In VV strain Copenhagen this was called A41L (Goebel et al., 1990), and in VV strain WR it was originally named SalF4L (Howard et al., 1991) but renamed SalF3L (Smith et al., 1991). Hereafter it is referred to as A41L. The relatedness of A41L and SFV T1, including the co-alignment of 8 cysteine residues was noted (Howard et al., 1991). Howard et al., 1991 also noted a potential site for attachment of carbohydrate via an asparagine residue and a putative signal peptide that might translocate the protein across the endoplasmic reticulum membrane and out of the cell. Other authors claimed there was no similarity between A41L and the 35 kDa protein of VV Lister and SFV T1 protein (Martinez-Pomares et al., 1995). In all three strains of variola major virus that have been sequenced there is an open reading frame (ORF) with more than 95% amino acid identity to the VV WR A41L protein termed 16L in strain Harvey (Aguado et al., 1992), A44L in strain Bangladesh-1975 (Massung et al., 1994) and A46L in strain India-1967 (Shchelkunov et al., 1994).
The similarity of A41L to vCKBP suggested that the A41L protein might bind chemokines as for vCKBP (Alcami et al., 1998). However Alcami et al., (1998) reported the supernatants of cells infected with VV WR or VV Lister from which the gene encoding the 35 kDa protein had been deleted (Patel et al., 1990), did not contain a protein that could be crosslinked to MIP-1&agr;, RANTES, IL-8 or GRO-&agr; (Alcami e

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